1. Field of the Invention
The invention relates to a method for measuring the concentration of a measuring object, a concentration measuring kit, and a sensor chip for use in the method. Particularly, the invention relates to a coloring reagent employed in an enzyme reaction for rapidly measuring the concentration of a measuring object using a very small quantity of a sample with high sensitivity and high accuracy by taking advantage of an evanescent wave, a concentration measuring kit, a method for measuring the concentration of a measuring object, and a sensor chip for use in the method.
2. Description of the Related Art
An enzyme-linked immunosorbent assay (ELISA) method has been practically used in clinical examinations as a method for measuring a very small quantity of a component by taking advantage of a specific reaction between an antigen and antibody.
A resin plate having 96 pits (wells) usually called as a microplate is used for the ELISA method. For example, a primary antibody is immobilized on each well depending on the object of assay in a sandwich ELISA method. In this method, a test sample solution is dispensed in each well, and the antibody (primary antibody) immobilized on the plate is allowed to react with a measuring object in the test sample solution (referred to a primary reaction hereinafter) followed by removing a solution containing the unreacted test sample by washing. Then, a solution of a secondary antibody labeled with an enzyme is dispensed in each well of the plate to permit the measuring object reacted with the secondary antibody to specifically react with the measuring object (referred to a secondary reaction hereinafter). After removing the solution of the unreacted secondary antibody, a solution of a coloring reagent is dispensed in each well for permitting an enzyme reaction to proceed (referred to an enzyme reaction hereinafter) to allow the enzyme reaction product to develop a color, and the concentration of the measuring object is determined from a calibration curve by measuring absorbance from the intensity of transmitted light through the well using a microplate reader.
For example, insulin is a hormone secreted from β-cells of the pancreas, and is known to have an action for decreasing the blood glucose level. Accordingly, the concentration of insulin in the blood should be measured for diagnosis of diabetes and for recognizing the pathology of the patient. The insulin concentration is measured by the same method as described above by ELISA using a microplate having wells in which an anti-insulin antibody is immobilized by dispensing a test sample solution in this well to allow the anti-insulin antibody to react with insulin in the test sample solution.
However, an amount of the test sample of several tens of microliters to 100 microliters is required for ELISA using the microplate, or 5 μL or more at the smallest, and sensitivity of the measurement reduces in the order of only about several hundreds of picograms per mL at a concentration described above. In addition, the reaction system is affected by an increased amount of inhibitory substances for the antigen-antibody reaction involved in the test sample solution when the amount of the test sample subjected to the measurement is increased in order to enhance sensitivity. Since sensitivity of the measurement may be rather decreased by increasing the amount of the test sample, it remains to be only about several hundreds of picograms per mL.
It is another problem of ELISA that the measurement takes a long time since the reaction time becomes long before the antigen-antibody reaction is completed in ELISA which requires a large amount of the test sample. For example, the time required for the primary reaction is usually several hours, or 24 hours at the longest, and the secondary reaction and substrate reaction require several tens minutes.
The amount of the test sample (blood, plasma) is desirably as small as possible when the test sample is collected from an infant or a small animal. While the amount of the test sample dispensed in the wells of the microplate is required to be accurate in ELISA, it is difficult to accurately measure an amount as small as less than 5 μL. Accordingly, the test sample in an amount of more than necessary should be sampled for accurate measurements, although it is desirable to subject a smaller amount of the sample to the measurement.
A sensor chip taking advantage of the antigen-antibody reaction has been known. FIG. 1 is a schematic illustration showing the construction of a sensor chip having an optical waveguide. The sensor chip comprises an optical waveguide layer 1 made of a silicon nitride film formed on a glass base plate 16, a pair of an incident side grating (diffraction grating) 13a and outgoing side grating 13b, or prisms (not shown), disposed at both sides of the optical waveguide layer, respectively, and an antibody immobilized layer 14 formed on the optical waveguide layer 1.
An antigen-antibody reaction occurs by allowing a test sample solution containing an antigen to contact the antibody immobilized layer 14 in such a sensor chip. An immune complex comprising antibody/antigen/fluorescent pigment-labeled antibody is formed on the base plate by adding a fluorescent pigment-labeled antibody solution in the antigen-antibody reaction system. The amount of the antigen in the test sample solution is assayed by the steps comprising: allowing a laser light to impinge through the incident side grating 13a into the optical waveguide layer 1 to emit an evanescent wave; exciting the fluorescent pigment by the evanescent wave in the antibody immobilized layer 14 on the optical waveguide layer 1; and analyzing the amount of the antigen in the test sample solution by detecting the intensity of fluorescence emitted from the fluorescent pigment with a photo-acceptance element (see, for example, Japanese Patent Application Laid-Open (JP-A) No. 8-285851).
The evanescent wave refers to an electromagnetic wave localizing at near an interface where a light is totally reflected at the interface between the optical waveguide layer and an external layer. Known measuring methods using the evanescent wave include a method for detecting a change in a physical value of the reflection light due to absorption of the evanescent wave in a pigment-labeled substance (for example a pigment-labeled secondary antibody) in the test sample, in addition to the method for labeling the test sample with the fluorescent pigment (see, for example, Jpn. Pat. Appln. KOKOKU Publication No. 3-7270).
In these conventional measuring methods, however, the number of the pigments or fluorescent pigments incorporated into the immune complex becomes small to make the measurement difficult when the concentration of the measuring object in the test sample is low. Accordingly, the photo-acceptance element for detecting the changes of the physical value of the reflection light should be highly sensitive to render the element to be expensive while the apparatus for detecting the changes of the physical value of the reflection light also becomes complicated and expansive.